Objectives: We estimated the association between bulky DNA adduct levels and birth weight in a multicenter study and examined modification of this association by maternal intake of fruits and vegetables during pregnancy.

Methods: Pregnant women from Denmark, England, Greece, Norway, and Spain were recruited in 2006–2010. Adduct levels were measured by the 32P-postlabeling technique in white blood cells from 229 mothers and 612 newborns. Maternal diet was examined through questionnaires.

We thank participants in the study and the collaborators from the hospitals.

The NewGeneris (Newborns and Genotoxic exposure risks) study was funded by the European Union (EU contract FOOD-CT-2005-016320). The study was also supported by the National Institute for Health Research, United Kingdom (programme grant RP-PG-0407-10044), the Norwegian Ministry of Health, the Norwegian Ministry of Education and Research, the Norwegian Research Council/FUGE (grant 151918/S10), the EU funded HiWATE (contract Food-CT-2006-036224), the U.S. National Institutes of Health (NIH)/National Institute of Environmental Health Sciences (contract NO-ES-75558), and the U.S. NIH/National Institute of Neurological Disorders and Stroke (grant 1 UO1 NS 047537-01). M.P. holds a Juan de la Cierva postdoctoral fellowship awarded from the Spanish Ministry of Science and Innovation (JCI-2011-09479).

The authors declare they have no actual or potential competing financial interests.

Introduction

Environmental exposures in utero may have adverse effects on health both immediately and in later life. Measurement of biomarkers in cord blood improves exposure assessment and may improve our understanding of biological mechanisms during this critical window of exposure and vulnerability (Wild and Kleinjans 2003).

Food is an important source of PAHs (IARC 2010; Kazerouni et al. 2001). Intake of meat with a blackened surface (Pedersen et al. 2012a), exposure to traffic-related air pollution during pregnancy (Pedersen et al. 2009), and smoking during pregnancy (Godschalk et al. 2005; Hansen et al. 1993; Pedersen et al. 2009) have been associated with higher levels of bulky DNA adducts in human cord blood. However, evidence regarding associations between bulky DNA adducts and birth outcomes is conflicting. Smoking-related DNA adducts measured by the 32P-postlabeling method in placental tissue from 30 women in the United States were associated with reduced birth weight (Everson et al. 1988), but in a study of 199 women in the Czech Republic, there were no associations between bulky DNA adducts in placenta tissue and birth weight, the risk of low birth weight (< 2,500 g), gestational duration, or preterm delivery (Sram et al. 2006).

Consumption of fruits and vegetables is considered beneficial for health (Slavin and Lloyd 2012) and may protect against cancer (World Cancer Research Fund/American Institute for Cancer Research 2007) through antioxidative effects and other properties related to dietary intake of fiber, folate, and other beneficial nutrients. In the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort study, higher intake of fibers was negatively associated with bulky DNA adduct levels in white blood cells from 1,085 adults (Peluso et al. 2008). In addition, a diet rich in vitamin C has been associated with lower levels of DNA damage [reviewed by Sram et al. (2012)], and high maternal vitamin C intake during pregnancy appeared to reduce the association between estimated maternal dietary benzo[a]pyrene (BaP) intake and size at birth of 586 newborns from Spain (Duarte-Salles et al. 2012).

In the present study, we investigated the association between bulky DNA adduct levels and birth weight in 612 newborns and further assessed whether maternal consumption of fruits and vegetables during pregnancy modified this association.

Methods

Study population. The study was conducted as a part of the NewGeneris (Newborns and Genotoxic exposure risks) study of the impact of diet during pregnancy on child health (Merlo et al. 2009). Pregnant women were enrolled during 2006–2010 from 11 maternity units located in Copenhagen, Denmark; Bradford, England; Heraklion, Greece; Oslo, Norway; and Barcelona and Sabadell, Spain (Pedersen et al. 2012b). Births were included in the present analysis if they occurred during the periods of cord blood collection and processing, if there was a sufficient volume of cord blood, and if blood processing and biomarker analysis was successful. Precise participation rates for the present analysis cannot be estimated because the number of births that might have been eligible cannot be determined.

Detailed information on personal characteristics was obtained from the mothers by using extensive questionnaires collected before or around the time of delivery (Table 1). The questionnaires were self-administered (Denmark-2009, Norway), partly supported (Denmark-2007, Spain, and England), and administered by an interviewer (Greece) (Pedersen et al. 2012b). Dietary information concerning diet during pregnancy was obtained from country-specific food frequency questionnaires. Information on birth weight, birth head circumference, gestational age, infant sex, and mode of delivery was obtained from maternity records. Gestational age at birth was based on last menstrual period and/or ultrasound-based estimated date of conception and corrected by ultrasound measurements if there was a discordance of ≥ 7 days between both estimates.

DNA was isolated centrally from ~ 0.5-mL aliquots of buffy-coat samples using Qiagen Midi Kit catalog no.13343 (Qiagen, Hilden, Germany) with some modifications (Arab et al. 2009). Levels of bulky DNA adducts were determined by using the 32P-postlabeling method with the nuclease P1 adduct enrichment version according to standardized protocols (Godschalk et al. 2005; Karttunen et al. 2010; Kovács et al. 2011). The protocols were harmonized and adjusted in an interlaboratory comparison study among the three 32P-postlabeling investigator laboratories, including the use of the same external benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)–DNA standard [111 adducts in 108 normal nucleotides (nt), which was a kind gift from F.F. Beland (National Center for Toxicological Research, Little Rock, AR, USA)]. All samples from Greece, Spain, and Norway and the Danish samples collected in 2006–2007 were analyzed in Budapest, Hungary (61% of the samples); the Danish samples from 2009 were analyzed in Stockholm, Sweden (21%); and the samples from England were analyzed in Maastricht, the Netherlands (18%). The interlaboratory comparison study showed a very high repeatability between two of the three laboratories, whereas the adduct levels measured in the third laboratory were consistently 3.7 times lower than the mean levels determined by the two other laboratories. Differences in DNA adduct determinations between laboratories normally occur because of the complicated multistep and sensitive procedures used for the detection of the adducts, and interlaboratory studies are therefore necessary. A correction for the laboratory factor of 3.7 was thus applied to the samples analyzed in Stockholm (Denmark-2009, 21% of the total). A sensitivity analysis that included or subsequently excluded these samples gave similar results, so all analyses were based on the total study population including the corrected data.

The individual level of DNA adducts was obtained as the average of at least two independent measurements. The detection limit of the assay was approximately 0.1–0.3 adducts per 108 unmodified nucleotides (n/108 nt).

We examined the effect of different degrees of adjustment for potential confounders on the association of bulky DNA adduct levels on birth weight. Potential confounders selected a priori for the adjusted model were gestational age (completed weeks, continuous), infant sex, maternal prepregnancy body mass index (BMI; kilograms per meter squared), parity (0, ≥ 1), maternal age (years), maternal ethnicity (white, nonwhite), self-reported maternal active smoking at the end of pregnancy (no, yes), self-reported maternal exposure to secondhand smoke (SHS) during pregnancy (no, yes), ethylene oxide–hemoglobin (Hb) adduct levels in cord blood (picomoles per gram Hb; to assess exposure to tobacco smoke during pregnancy), mode of delivery (vaginal, cesarean section), dietary supplements (none, any), maternal consumption of fruits and vegetables during pregnancy (low, high), season of delivery (March–May, June–August, September–November, December–February), country of delivery, and maternal education (low, middle, high) as a marker of socioeconomic position. In addition, we estimated basic adjusted associations using models that included country, maternal smoking at the end of pregnancy, sex, and gestational age only.

Given that bulky DNA adduct levels in cord blood reflect a steady state between DNA damage and repair during the previous few months (Godschalk et al. 2005), we classified women who smoked during the last four months of pregnancy as smokers, whereas those who never smoked or who quit before the last 4 months of pregnancy were classified as nonsmokers. Women exposed to SHS in the home and/or elsewhere during pregnancy were categorized as exposed. In addition, to further assess exposure to dietary acrylamide and tobacco smoke during pregnancy, we adjusted for acrylamide and ethylene oxide Hb adduct levels (picomoles per gram Hb), respectively, in cord blood samples (von Stedingk et al. 2011).

Maternal intake of fruits and vegetables during pregnancy (grams/day, based on 20–61 questionnaire items depending on country) was categorized as high or low according to overall and country-specific median levels as a proxy measure of the consumption of nutrients that might be protective against genotoxic activation processes. We also classified mothers according to their intake of fruits high in vitamin C and other antioxidants (i.e., all types of citrus fruits, both in terms of whole fruits and juice, kiwi fruit, and berries, ranging from three to seven questionnaire items). Women (n = 54) with a total energy estimate of < 500 or > 6,000 kcal/day were excluded from adjusted analyses and analyses of effect modification by diet (Butte and King 2005; Willett 1998).

In addition, we performed a meta-analysis by country to derive country-specific effect estimates of associations between cord blood DNA adduct levels and birth weight. Pregnancy outcomes in Northern European countries (England, Denmark, and Norway, n = 367), which had low average levels of adducts, were compared with outcomes in Southern European countries (Greece and Spain, n = 245), which had higher average adduct levels.

To estimate the association between bulky DNA adduct levels and term birth weight, we repeated the main analysis after excluding preterm deliveries (n = 26). We used an alpha level of 5% for statistical significance. All statistical analyses were performed using Stata S.E. version 12.1 (StataCorp, College Station, TX, USA).

Results

Study population. The study population was composed of neonates from Denmark (33%), Spain (29%), England (18%), Greece (11%), and Norway (10%). Mothers were predominantly white Europeans, multiparous, and nonsmoking (Table 1). Most children were born at term (96%) and weighed > 2,500 g at birth (97%). Some study population characteristics differed significantly between the Northern and Southern European populations (e.g., maternal smoking; 8% vs. 18%, respectively), whereas characteristics such as maternal prepregnancy BMI and dietary supplement use were similar. The daily median fruit and vegetable intake of the Southern European mothers was 58 g higher than that of Northern European mothers, but the difference was not statically significant (p = 0.13). The difference in fruit and vegetable intake between Southern and Northern European mothers was also not statically significant after adjustment for individual total energy intake. Differences were smaller for fruits high in vitamin C (Table 1).

Figure 1 – Birth weight (A–B) and bulky DNA adduct levels in cord blood (C–D):­distribution by country (A,C) and region (B,D). The horizontal line is the median, and the bottom and top of the box are the first and third quartiles. Whiskers indicate the variability outside the upper and lower quartiles (i.e., within 1.5 interquartile range of the lower quartile and upper quartile). Outliers are plotted as individual dots.

The median bulky DNA adduct level in the 71 children born to mothers who actively smoked at the end of their pregnancy (10.9 adducts/108 nt; range, 0.6–73.9) was higher than the median level in the 541 children of mothers who never smoked or who quit before the last 4 months of pregnancy (8.2 adducts/108 nt; range, 0.6–87.5, p = 0.07). Adduct levels were lowest in the 505 children born to mothers who never smoked during their pregnancy (8.0 adducts/108 nt; range, 0.6–87.5, p = 0.10).

Adduct levels in cord blood and birth outcomes. Higher levels of bulky DNA adducts in cord blood were associated with lower birth weight (Table 2). For the full study population (n = 612), the estimated difference in mean birth weight for infants in the highest tertile versus the lowest tertile of adduct levels was –110 g (95% CI: –192, –28 g), based on basic adjusted models that included country, maternal smoking at the end of pregnancy, sex, and gestational age only. The corresponding association was similar when restricted to the 541 mothers who did not smoke during the last 4 months of pregnancy (–108 g; 95% CI: –202, –14 g), but was slightly stronger when restricted to the 505 mothers who did not smoke at any time during the pregnancy (–124 g; 95% CI: –216, –32 g).

Differences between countries. Consistent with associations estimated for the full study population, mean birth weight was significantly lower for Northern European infants in the highest versus lowest tertile of adduct levels (–119 g; 95% CI: –234, –4 g) based on the basic adjusted model. However, there was a nonsignificant positive association among the 245 Southern Europeans (71 g; 95% CI: –59, 202 g; p = 0.28). Estimates based on adjusted models also indicated negative associations for the Northern European countries, but not for the Southern European countries (Figure 2; p-value for heterogeneity = 0.03), and when imputed data were used to include individuals with missing information on covariates (results not shown). Therefore, although birth weight was lowest and adduct levels were highest in Greece and Spain (Figure 1), the negative association between adduct levels and birth weight estimated for the full study population appears to be driven by the Northern countries.

Figure 2 – Change in birth weight (g) associated with the bulky DNA adduct levels in cord blood (per 108 nt) by country. Country-specific effect estimates (ES) and their 95% CIs as well as the meta-analyses combined effect estimates (random effect of country), which correspond to the change in birth weight for the highest relative to the lowest tertile of cord blood bulky DNA adduct levels further adjusted as described in Table 2 (n = 409). Black diamonds indicate ESs; gray squares are proportional to the country-specific weights used in the meta-analyses, and the associated 95% CIs are shown as horizontal black lines. The summary ES, which corresponds to the change in birth weight (g), is indicated with a red dashed vertical line and blue diamond, and the associated 95% CIs are indicated by the lateral tips of the diamond. The solid vertical black line indicates no change in birth weight.

Differences by intake of fruits and vegetables. The association of bulky DNA adduct levels with birth weight differed according to maternal intake of fruits and vegetables and intake of fruits high in vitamin C, although interactions were only marginally significant (Table 3). The estimated difference in birth weight between the highest and lowest tertiles of adduct levels was greater among births to mothers with low intakes of fruits and vegetables (–248 g; 95% CI: –405, –92 g) than among births to mothers with high intakes (–58 g; 95% CI: –206, 90 g; p = 0.44). Consumption of dietary supplements during pregnancy was common in both Northern and Southern Europeans (85% and 90%, respectively) and did not appear to modify or confound associations between adduct levels and birth weight (data not shown).

Table 3 – Modification of the change in birth weight (g) associated with bulky DNA adduct levels by maternal intake of fruits and vegetables during pregnancy.

Discussion

We measured levels of bulky DNA adducts in white blood cells from cord blood in a large multicenter European prospective general population study and found that higher adduct levels in cord blood were significantly negatively associated with birth weight. The negative association was observed among newborns from England, Denmark, and Norway, who had the lowest average adduct levels, but was not evident among Southern Europeans, who had the highest mean adduct levels. The negative association with birth weight was stronger among the children of mothers with low versus high intakes of fruits and vegetables.

Our findings suggest that maternal intake of fruits and vegetables may modify the association between bulky DNA adduct levels and birth weight. This finding is consistent with the findings of a questionnaire-based study evaluating dietary BaP (Duarte-Salles et al. 2012).

Children from Southern Europe had, on average, higher bulky DNA adduct levels and lower birth weight than Northern European children. However a negative association between adducts and birth weight was found only in the Northern Europeans. The same pattern of higher bulky DNA adduct levels in Southern Europe than in Northern Europe has been found in adults (Ricceri et al. 2010), and this may reflect differences in ambient air quality (Table 4), but also wider geographical differences in diet, food preparations, and other factors, or perhaps different susceptibility toward environmental genotoxic agents. It is possible that exposures that cause adducts in children from Northern Europe also cause reduced birth weight, whereas exposures responsible for adducts among Southern European children might differ and may not affect birth weight. A complementary explanation could involve a saturation effect of the toxicity of these adducts on birth weight, although other studies in populations exposed to very high air pollution levels and corresponding high adduct levels have reported negative associations with birth weight (Perera et al. 1998, 2005).

A key strength of the present study is the measurement of bulky DNA adducts in cord blood, which enabled a more accurate evaluation of the biologically effective dose of genotoxic agents resulting from complex environmental exposures than estimates based solely on external exposure assessment. Large biomarker-based studies are rare because of their costs and complexity. Standardized protocols were developed (Merlo et al. 2009) and applied to the collection of cord blood from multiple study centers. Detailed information on maternal characteristics and diet was collected in a manner that allowed pooling of data from five different countries (Pedersen et al. 2012b). In addition to increasing the sample size, enrolling participants from different countries allows us to test hypotheses in different settings.

32P-Postlabeling is considered to be the most suitable and sensitive method for assessing total DNA damage resulting from exposures to unknown, complex mixtures of genotoxic compounds. We used the validated in vitro BPDE–DNA standard in each 32P-postlabeling session in duplicate for normalization of the DNA adduct levels. Each DNA sample was analyzed at least twice. The interlaboratory comparison study, the use of common protocols, and use of a validated BPDE–DNA standard minimized the potential for measurement error. Thus, we believe that measurement error of the method was properly handled and is not a special concern in our study.

Low birth weight is an important outcome because it is associated with greater risk of neonatal mortality, hypertension and cardiovascular disease, diabetes, certain cancers, reduced and/or delayed postnatal growth, and cognitive development (Gluckman et al. 2008). Our findings suggest that environmental exposures that result in the in utero formation of bulky DNA adducts also may affect prenatal growth, and that this potential effect may be reduced by high maternal fruit and vegetable consumption.

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